The complement system, an immune response effector mechanism, comprises a set of proteins activated by the presence of antibody/antigen complexes in the host to initiate a cascade of proteolytic cleavage and protein binding events which promote elimination of the antigens from the host. Physiological responses to activation of the complement system include acute local inflammation, dilation of blood vessels, and transudation of fluid into interstitial spaces.
The human complement system includes component C1, a complex of protein subcomponents including serine esterase (C1-esterase), which participates in the proteolytic cleavage sequence of the mechanism. Activation of the C1 component of the complement system and enzymatic activity of C1-esterase is regulated by a serum inhibitor, C1-inactivator (C1-inhibitor) which controls the activity of C1-esterase. A functional deficiency of this inactivator protein results in a condition termed hereditary angioneurotic edema, caused by repeated unchecked activation of the complement system and symptomized by recurrent episodes of local acute inflammation at sites of activation. The accompanying vessel dilation and transudation of fluid into tissue spaces can cause suffocation if activation occurs at sensitive sites in the upper respiratory tract.
Acute attacks of swelling in hereditary angioedema are successfully treated by the administration of functionally active serum C1-inactivator (C1-INA), preferably as a concentrated plasma fraction. Plasma concentrates of C1-INA currently used in clinical replacement therapy have been implicated as a cause of transfusion hepatitis, however. Accordingly, research efforts have been directed to methods for preparing C1-INA concentrates which provide a high yield of biologically active C1-INA and which also provide a product concentrate having minimized potential for transmitting hepatitis B virus (herein also referred to as HBV).
Current methods for purification of C1-INA typically include the step of polyethylene glycol (PEG) precipitation, which removes extraneous complement system proteins; the product C1-INA fraction, however, retains potential for viral infection. The classic pasteurization approach to inactivation of plasma HBV, successfully employed in providing safe clinical serum albumin, has not heretofore been useful in providing safe clinical C1-INA, since this protein denatures under the required pasteurization conditions. Attempts to improve the heat stability of C1-INA have not significantly improved recovery of active material. The use of N-acetyltryptophanate, sodium caprylate, and sodium citrate, successfully employed as heat stabilizers in the pasteurization of the plasma proteins albumin and antithrombin III, respectively, have not provided adequate protection for C1-INA during pasteurization. Losses in C1-INA activity attributable to heat denaturation during pasteurization in the presence of sodium citrate, for example, typically exceed 25%; in addition to lacking cost-effectiveness, the large amount of protein denatured and subsequently infused into patients can adversely affect continued efficacy of the treatment. Heat denaturation has been encountered in the pasteurization of other plasma proteins such as Factor IX, used for replacement therapy in the treatment of hemophilia B.